Nucleotide sequences which encode the pfk gene

ABSTRACT

The present invention is directed to nucleotide sequences coding for phosphofructokinase which have been  Corynebacterium glutamicum , and a process for the production of an L-amino acid comprising culturing a coryneform bacteria comprising an overexpressed pfk gene, wherein said pfk gene comprises a polynucleotide having a nucleotide sequence which is at least 90% identical to the nucleotide of SEQ ID NO: 1 encoding a polypeptide having the enzymatic activity of a phosphofructokinase, accumulating said L-amino acid in the medium or in the cells of said bacterium, and isolated said L-amino acid.

This application claims priority from German Application No. 199 56131.1, filed on Nov. 23, 1999, the subject matter of which is herebyincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention provides nucleotide sequences which encode the pfk geneand a process for the fermentative preparation of amino acids, inparticular L-lysine, using coryneform bacteria in which the pfk gene isenhanced.

2. Background Information

Amino acids, in particular L-lysine, are used in human medicine and inthe pharmaceuticals industry, but in particular in animal nutrition.

It is known that amino acids are prepared by fermentation from strainsof coryneform bacteria, in particular Corynebacterium glutamicum.Because of their great importance, work is constantly being undertakento improve the preparation processes. Improvements to the processes canrelate to fermentation measures, such as, for example, stirring andsupply of oxygen, or the composition of the nutrient media, such as, forexample, the sugar concentration during the fermentation, or the workingup to the product form by, for example, ion exchange chromatography, orthe intrinsic output properties of the microorganism itself.

Methods of mutagenesis, selection and mutant selection are used toimprove the output properties of these microorganisms. Strains which areresistant to antimetabolites, such as, for example, the lysine analogueS-(2-aminoethyl)-cysteine, or are auxotrophic for metabolites ofregulatory importance and produce L-amino acids, such as, for example,L-lysine, are obtained in this manner.

Recombinant DNA techniques have also been employed for some years forimproving Corynebacterium strains which produce amino acids, byamplifying individual amino acid biosynthesis genes and investigatingthe effects of such changes on the amino acid production. Reviewarticles on this subject are to be found, inter alia, in Kinoshita(“Glutamic Acid Bacteria”, in: Biology of Industrial Microorganisms,Demain and Solomon (Eds.), Benjamin Cummings, London, UK, 1985,115-142), Hilliger (BioTec 2, 40-44 (1991)), Eggeling (Amino Acids6:261-272 (1994)), Jetten and Sinskey (Critical Reviews in Biotechnology15, 73-103 (1995)) and Sahm et al. (Annuals of the New York Academy ofScience 782, 25-39 (1996)).

SUMMARY OF THE INVENTION Object of the Invention

It is an object of the invention to provide new means for improvedfermentative preparation of amino acids, in particular L-lysine.

Description of the Invention

Amino acids, in particular L-lysine, are used in human medicine, in thepharmaceuticals industry and in particular in animal nutrition. There istherefore a general interest in providing new improved processes for thepreparation of amino acids, in particular L-lysine.

When L-lysine or lysine are mentioned in the following, not only thebase but also the salts, such as, for example, lysine monohydrochlorideor lysine sulfate, are also meant. The invention provides an isolatedpolynucleotide from coryneform bacteria, comprising a polynucleotidesequence chosen from the group consisting of

-   a) a polynucleotide which is at least 70% identical to a    polynucleotide which encodes a polypeptide which comprises the amino    acid sequence of SEQ ID NO:2,-   b) a polynucleotide which encodes a polypeptide which comprises an    amino acid sequence which is at least 70% identical to the amino    acid sequence of SEQ ID NO:2,-   c) a polynucleotide which is complementary to the polynucleotides    of a) or b), and-   d) a polynucleotide comprising at least 15 successive nucleotides of    the polynucleotide sequence of a), b) or c).

In a preferred embodiment, the invention also provides thepolynucleotide with the features described above, preferably being a DNAwhich is capable of replication, comprising:

-   (i) the nucleotide sequence shown in SEQ ID NO:1, or-   (ii) at least one sequence which corresponds to sequence (i) within    the range of the degeneration of the genetic code, or-   (iii) at least one sequence which hybridizes with the sequence    complementary to sequence (i) or (ii), and optionally-   (iv) sense mutations of neutral function in (i).

The invention also provides

-   a polynucleotide with the aforementioned features, comprising the    nucleotide sequence as shown in SEQ ID NO:1,-   a polynucleotide with the aforementioned features, which encodes a    polypeptide which comprises the amino acid sequence as shown in SEQ    ID NO:2,-   a vector containing the polynucleotide with features a)-d) above, in    particular a shuttle vector or plasmid vector and coryneform    bacteria serving as the host cell, which contain the vector.

The invention also provides polynucleotides which substantially comprisea polynucleotide sequence, which are obtainable by screening by means ofhybridization of a corresponding gene library, which comprises thecomplete gene with the polynucleotide sequence corresponding to SEQ IDNO:1, with a probe which comprises the sequence of the polynucleotidementioned, according to SEQ ID no. 1 or a fragment thereof, andisolation of the DNA sequence mentioned.

Polynucleotide sequences according to the invention are suitable ashybridization probes for RNA, cDNA and DNA, in order to isolate, in thefull length, cDNA which code for phosphofructokinase and to isolatethose cDNA or genes which have a high similarity of sequence with thatof the phosphofructokinase gene.

Polynucleotide sequences according to the invention are furthermoresuitable as primers for the preparation of DNA of genes which code forphosphofructokinase by the polymerase chain reaction (PCR).

Such oligonucleotides which serve as probes or primers comprise at least30, preferably at least 20, very particularly preferably at least 15successive nucleotides.

Oligonucleotides which have a length of at least 40 or 50 nucleotidesare also suitable.

“Isolated” means separated from its natural environment.

“Polynucleotide” generally relates to polyribonucleotides andpolydeoxyribonucleotides, wherein the RNA or DNA may be modified orunmodified.

“Polypeptides” is understood as meaning peptides or proteins whichcomprise two or more amino acids bonded via peptide bonds.

The polypeptides according to the invention include a polypeptideaccording to SEQ ID NO:2, in particular those with the biologicalactivity of phosphofructokinase, and also those which are at least 70%identical to the polypeptide according to SEQ ID NO:2, and preferablyare at least 80% identical, and most preferably 90% to 95% identical tothe polypeptide according to SEQ ID NO:2 and have the activitymentioned.

The invention also provides a process for the fermentative preparationof amino acids, in particular L-lysine, using coryneform bacteria whichin particular already produce an amino acid, and in which the nucleotidesequences which code for the pfk gene are enhanced, in particularover-expressed.

The term “enhancement” in this connection describes the increase in theintracellular activity of one or more enzymes in a microorganism whichare encoded by the corresponding DNA, for example by increasing thenumber of copies of the gene or genes, using a potent promoter or usinga gene which encodes a corresponding enzyme having a high activity, andoptionally combining these measures.

The microorganisms provided by the present invention can be used toprepare L-amino acids, in particular L-lysine, from glucose, sucrose,lactose, fructose, maltose, molasses, starch, cellulose or from glyceroland ethanol. They can be representatives of coryneform bacteria, inparticular of the genus Corynebacterium. Of the genus Corynebacterium,the species Corynebacterium glutamicum, which is known among experts forits ability to produce L-amino acids, is particularly useful.

Suitable strains of the genus Corynebacterium, in particular of thespecies Corynebacterium glutamicum, are, for example, the knownwild-type strains

Corynebacterium glutamicum ATCC13032

Corynebacterium acetoglutamicum ATCC15806

Corynebacterium acetoacidophilum ATCC13870

Corynebacterium thermoaminogenes FERM BP-1539

Corynebacterium melassecola ATCC17965

Brevibacterium flavum ATCC14067

Brevibacterium lactofermentum ATCC13869 and

Brevibacterium divaricatum ATCC14020

and L-lysine-producing mutants or strains prepared therefrom, such as,for example

Corynebacterium glutamicum FERM-P 1709

Brevibacterium flavum FERM-P 1708

Brevibacterium lactofermentum FERM-P 1712

Corynebacterium glutamicum FERM-P 6463

Corynebacterium glutamicum FERM-P 6464 and

Corynebacterium glutamicum DSM5715.

The inventors have succeeded in isolating the new pfk gene of C.glutamicum which codes for the enzyme phosphofructokinase.

To isolate the pfk gene or also other genes of C. glutamicum, a genelibrary of this microorganism is first constructed in E. coli. Theconstruction of gene libraries is described in generally known textbooksand handbooks.

The textbook by Winnacker: Gene und Klone, Eine Einfuhrung in dieGentechnologie [Genes and Clones, An Introduction to GeneticEngineering] (Verlag Chemie, Weinheim, Germany, 1990) or the handbook bySambrook et al.: Molecular Cloning, A Laboratory Manual (Cold SpringHarbor Laboratory Press, 1989) may be mentioned as examples. Awell-known gene library is that of the E. coli K-12 strain W3110constructed in λ vectors by Kohara et al. (Cell 50, 495-508 (1987)).Bathe et al. (Molecular and General Genetics, 252:255-265, 1996)describe a gene library of C. glutamicum ATCC13032, which wasconstructed with the aid of the cosmid vector SuperCos I (Wahl et al.,1987, Proceedings of the National Academy of Sciences USA, 84:2160-2164)in the E. coli K-12 strain NMS54 (Raleigh et al., 1988, Nucleic AcidsResearch 16:1563-1575). Börmann et al. (Molecular Microbiology 6(3),317-326)) (1992)) in turn describe a gene library of C. glutamicumATCC13032 using the cosmid pHC79 (Hohn and Collins, Gene 11, 291-298(1980)). To prepare a gene library of C. glutamicum in E. coli it isalso possible to use plasmids such as pBR322 (Bolivar, Life Sciences,25, 807-818 (1979)) or pUC9 (Vieira et al., 1982, Gene, 19:259-268).Suitable hosts are, in particular, those E. coli strains which arerestriction- and recombination-defective. An example of these is thestrain DH5αmcr, which has been described by Grant et al. (Proceedings ofthe National Academy of Sciences USA, 87 (1990) 4645-4649). The long DNAfragments cloned with the aid of cosmids can then in turn be subclonedin the usual vector suitable for sequencing and then sequenced, as isdescribed, for example, by Sanger et al. (Proceedings of the NationalAcademy of Sciences of the United States of America, 74:5463-5467,1977).

The new DNA sequence of C. glutamicum which encodes the pfk gene andwhich, as SEQ ID NO:1, is a constituent of the present invention, wasobtained in this manner. The amino acid sequence of the correspondingprotein has furthermore been derived from the present DNA sequence bythe methods described above. The resulting amino acid sequence of thepfk gene product is shown in SEQ ID NO:2.

Coding DNA sequences which arise from SEQ ID NO:1 as a result of thedegeneracy of the genetic code are also a constituent of the invention.In the same way, DNA sequences which hybridize with SEQ ID NO:1 or partsof SEQ ID NO:1 are a constituent of the invention. Conservative aminoacid exchanges, such as, for example, the exchange of glycine foralanine or of aspartic acid for glutamic acid in proteins, arefurthermore known among experts as “sense mutations” which do not leadto a fundamental change in the activity of the protein, i.e. are ofneutral function. It is furthermore known that changes on the N and/or Cterminus of a protein cannot substantially impair or can even stabilizethe function thereof. Information in this context can be found by theexpert, inter alia, in Ben-Bassat et al. (Journal of Bacteriology169:751-757 (1987)), in O'Regan et al. (Gene 77:237-251 (1989)), inSahin-Toth et al. (Protein Sciences 3:240-247 (1994)), in Hochuli et al.(Bio/Technology 6:1321-1325 (1988)) and in known textbooks of geneticsand molecular biology. Amino acid sequences which result in acorresponding manner from SEQ ID NO:2 are also a constituent of theinvention.

In the same way, DNA sequences which hybridize with SEQ ID NO:1 orportions of SEQ ID NO:1 are a constituent of the invention. Finally, DNAsequences which are prepared by the polymerase chain reaction (PCR)using primers which result from SEQ ID NO:1 are a constituent of theinvention. Such oligonucleotides typically have a length of at least 15nucleotides.

Instructions for identifying DNA sequences by means of hybridization canbe found by the expert, inter alia, in the handbook “The DIG SystemUsers Guide for Filter Hybridization” from Boehringer Mannheim GmbH(Mannheim, Germany, 1993) and in Liebl et al. (International Journal ofSystematic Bacteriology (1991) 41: 255-260). Instructions foramplification of DNA sequences with the aid of the polymerase chainreaction (PCR) can be found by the expert, inter alia, in the handbookby Gait: Oligonucleotide synthesis: a practical approach (IRL Press,Oxford, UK, 1984) and in Newton and Graham: PCR (Spektrum AkademischerVerlag, Heidelberg, Germany, 1994).

The inventors have found that coryneform bacteria produce amino acids,in particular L-lysine, in an improved manner after over-expression ofthe pfk gene.

To achieve over-expression, the number of copies of the correspondinggenes can be increased, or the promoter and regulation region or theribosome binding site upstream of the structural gene can be mutated.Expression cassettes which are incorporated upstream of the structuralgene act in the same way. By inducible promoters, it is additionallypossible to increase the expression in the course of fermentativeL-lysine production. The expression is likewise improved by measures toprolong the life of the mRNA. Furthermore, the enzyme activity is alsoincreased by preventing the degradation of the enzyme protein. The genesor gene constructions can either be present in plasmids with a varyingnumber of copies, or can be integrated and amplified in the chromosome.Alternatively, an over-expression of the genes in question canfurthermore be achieved by changing the composition of the media and theculture procedure.

Instructions in this context can be found by the expert, inter alia, inMartin et al. (Bio/Technology 5, 137-146 (1987)), in Guerrero et al.(Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio/Technology 6,428-430 (1988)), in Eikmanns et al. (Gene 102, 93-98 (1991)), inEuropean Patent Specification EPS 0 472 869, in U.S. Pat. No. 4,601,893,in Schwarzer and Pühler (Bio/Technology 9, 84-87 (1991), in Reinscheidet al. (Applied and Environmental Microbiology 60, 126-132 (1994)), inLaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)), inPatent Application WO 96/15246, in Malumbres et al. (Gene 134, 15-24(1993)), in Japanese Laid-Open Specification JP-A-10-229891, in Jensenand Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)), inMakrides (Microbiological Reviews 60:512-538 (1996)) and in knowntextbooks of genetics and molecular biology.

By way of example, the pfk gene according to the invention wasover-expressed with the aid of plasmids.

Suitable plasmids are those which are replicated in coryneform bacteria.Numerous known plasmid vectors, such as, for example, pZ1 (Menkel etal., Applied and Environmental Microbiology (1989) 64: 549-554), pEKEx1(Eikmanns et al., Gene 102:93-98 (1991)) or pHS2-1 (Sonnen et al., Gene107:69-74 (1991)) are based on the cryptic plasmids pHM1519, pBL1 orpGA1. Other plasmid vectors, such as, for example, those based on pCG4(U.S. Pat. No. 4,489,160) or pNG2 (Serwold-Davis et al., FEMSMicrobiology Letters 66, 119-124 (1990)), or pAG1 (U.S. Pat. No.5,158,891) can be used in the same manner.

Plasmid vectors which are furthermore suitable are those with the aid ofwhich the process of gene amplification by integration into thechromosome can be used, as has been described, for example, byReinscheid et al. (Applied and Environmental Microbiology 60, 126-132(1994)) for duplication or amplification of the hom-thrB operon. In thismethod, the complete gene is cloned in a plasmid vector which canreplicate in a host (typically E. coli), but not in C. glutamicum.Possible vectors are, for example, pSUP301 (Simon et al., Bio/Technology1, 784-791 (1983)), pK18mob or pK19mob (Schafer et al., Gene 145, 69-73(1994)), pGEM-T (Promega corporation, Madison, Wis., USA), pCR2.1-TOPO(Shuman (1994). Journal of Biological Chemistry 269:32678-84; U.S. Pat.No. 5,487,993), pCR®Blunt (Invitrogen, Groningen, Holland; Bernard etal., Journal of Molecular Biology, 234: 534-541 (1993)) or pEM1(Schrumpf et al, 1991, Journal of Bacteriology 173:4510-4516). Theplasmid vector which contains the gene to be amplified is thentransferred into the desired strain of C. glutamicum by conjugation ortransformation. The method of conjugation is described, for example, bySchäfer et al. (Applied and Environmental Microbiology 60, 756-759(1994)). Methods for transformation are described, for example, byThierbach et al. (Applied Microbiology and Biotechnology 29, 356-362(1988)), Dunican and Shivnan (Bio/Technology 7, 1067-1070 (1989)) andTauch et al. (FEMS Microbiological Letters 123, 343-347 (1994)). Afterhomologous recombination by means of a “cross over” event, the resultingstrain contains at least two copies of the gene in question.

In addition, it may be advantageous for the production of amino acids,in particular L-lysine, to enhance or over-express one or more enzymesof the particular biosynthesis pathway, of glycolysis, of anaplerosis,of the citric acid cycle or of amino acid export, in addition to the pfkgene.

Thus, for example, for the preparation of L-lysine, one or more geneschosen from the group consisting of

-   -   the dapA gene which encodes dihydrodipicolinate synthase (EP-B 0        197 335), or    -   the gap gene which encodes glyceraldehyde 3-phosphate        dehydrogenase (Eikmanns (1992), Journal of Bacteriology        174:6076-6086), or    -   the tpi gene which encodes triose phosphate isomerase (Eikmanns        (1992), Journal of Bacteriology 174:6076-6086), or    -   the pgk gene which encodes 3-phosphoglycerate kinase (Eikmanns        (1992), Journal of Bacteriology 174:6076-6086), or    -   the pyc gene which encodes pyruvate carboxylase (Eikmanns        (1992), Journal of Bacteriology 174:6076-6086), or    -   the lysE gene which encodes lysine export (DE-A-195 48 222)        can be over-expressed at the same time.

For the production of amino acids, in particular L-lysine, it mayfurthermore be advantageous to attenuate, in addition to the pfk gene,

-   -   the pck gene which encodes phosphoenol pyruvate carboxykinase        (DE 199 50 409.1, DSM 13047) and/or    -   the pgi gene which encodes glucose 6-phosphate isomerase (U.S.        Ser. No. 09/396,478, DSM 12969)        at the same time.

In addition to over-expression of the pfk gene it may furthermore beadvantageous, for the production of amino acids, in particular L-lysine,to eliminate undesirable side reactions, (Nakayama: “Breeding of AminoAcid Producing Micro-organisms”, in: Overproduction of MicrobialProducts, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK,1982).

The microorganisms prepared according to the invention can be culturedcontinuously or discontinuously in the batch process (batch culture) orin the fed batch (feed process) or repeated fed batch process(repetitive feed process) for the purpose of production of amino acids,in particular L-lysine. A summary of known culture methods is containedin the textbook by Chmiel (Bioprozesstechnik 1. Einführung in dieBioverfahrenstechnik [Bioprocess Technology 1. Introduction toBioprocess Technology (Gustav Fischer Verlag, Stuttgart, 1991)) or inthe textbook by Storhas (Bioreaktoren und periphere Einrichtungen[Bioreactors and Peripheral Equipment] (Vieweg Verlag,Braunschweig/Wiesbaden, 1994)).

The culture medium to be used must meet the requirements of theparticular strains in a suitable manner. Descriptions of culture mediafor various microorganisms are contained in the handbook “Manual ofMethods for General Bacteriology” of the American Society forBacteriology (Washington D.C., USA, 1981). Sugars and carbohydrates,such as, for example, glucose, sucrose, lactose, fructose, maltose,molasses, starch and cellulose, oils and fats, such as, for example,soya oil, sunflower oil, groundnut oil and coconut fat, fatty acids,such as, for example, palmitic acid, stearic acid and linoleic acid,alcohols, such as, for example, glycerol and ethanol, and organic acids,such as, for example, acetic acid, can be used as the source of carbon.These substances can be used individually or as a mixture. Organicnitrogen-containing compounds, such as peptones, yeast extract, meatextract, malt extract, corn steep liquor, soya bean flour and urea, orinorganic compounds, such as ammonium sulfate, ammonium chloride,ammonium phosphate, ammonium carbonate and ammonium nitrate, can be usedas the source of nitrogen. The sources of nitrogen can be usedindividually or as a mixture. Phosphoric acid, potassium dihydrogenphosphate or dipotassium hydrogen phosphate or the correspondingsodium-containing salts can be used as the source of phosphorus. Theculture medium must furthermore comprise salts of metals, such as, forexample, magnesium sulfate or iron sulfate, which are necessary forgrowth. Finally, essential growth substances, such as amino acids andvitamins, can be employed in addition to the abovementioned substances.Suitable precursors can moreover be added to the culture medium. Thestarting substances mentioned can be added to the culture in the form ofa single batch, or can be fed in during the culture in a suitablemanner.

Basic compounds, such as sodium hydroxide, potassium hydroxide, ammoniaor aqueous ammonia, or acid compounds, such as phosphoric acid orsulfuric acid, can be employed in a suitable manner to control the pH.Antifoams, such as, for example, fatty acid polyglycol esters, can beemployed to control the development of foam. Suitable substances havinga selective action, such as, for example, antibiotics, can be added tothe medium to maintain the stability of plasmids. To maintain aerobicconditions, oxygen or oxygen-containing gas mixtures, such as, forexample, air, are introduced into the culture. The temperature of theculture is usually 20° C. to 45° C., and preferably 25° C. to 40° C.Culturing is continued until a maximum of lysine has formed. This targetis usually reached within 10 hours to 160 hours.

The analysis of L-lysine can be carried out by anion exchangechromatography with subsequent ninhydrin derivatization, as described bySpackman et al. (Analytical Chemistry, 30, (1958), 1190).

The process according to the invention is used for the fermentativepreparation of amino acids, in particular L-lysine.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1: Plasmid pZ-pfkex

The abbreviations used in the figure have the following meaning:

Kan: Resistance gene for kanamycin

Ptac: tac promoter

pfk: pfk gene of C. glutamicum

rrnB-T1T2: Terminator T1T2 of the rrnB gene of E. coli

rep: Plasmid-coded replication origin for C. glutamicum (of pHM1519)

EcoRI: Cleavage site of the restriction enzyme EcoRI

SalI: cleavage site of the restriction enzyme SalI

DETAILED DESCRIPTION OF THE INVENTION

The present invention is explained in more detail in the following withthe aid of embodiment examples.

Example 1

Preparation of a Genomic Cosmid Gene Library from Corynebacteriumglutamicum ATCC 13032

Chromosomal DNA from Corynebacterium glutamicum ATCC 13032 was isolatedas described by Tauch et al. (1995, Plasmid 33:168-179) and partlycleaved with the restriction enzyme Sau3AI (Amersham Pharmacia,Freiburg, Germany, Product Description Sau3AI, Code no. 27-0913-O₂). TheDNA fragments were dephosphorylated with shrimp alkaline phosphatase(Roche Molecular Biochemicals, Mannheim, Germany, Product DescriptionSAP, Code no. 1758250). The DNA of the cosmid vector SuperCos1 (Wahl etal. (1987) Proceedings of the National Academy of Sciences USA84:2160-2164), obtained from the company Stratagene (La Jolla, USA,Product Description SuperCos1 Cosmid Vektor Kit, Code no. 251301) wascleaved with the restriction enzyme XbaI (Amersham Pharmacia, Freiburg,Germany, Product Description XbaI, Code no. 27-0948-O₂) and likewisedephosphorylated with shrimp alkaline phosphatase. The cosmid DNA wasthen cleaved with the restriction enzyme BamHI (Amersham Pharmacia,Freiburg, Germany, Product Description BamHI, Code no. 27-0868-04). Thecosmid DNA treated in this manner was mixed with the treated ATCC13032DNA and the batch was treated with T4 DNA ligase (Amersham Pharmacia,Freiburg, Germany, Product Description T4-DNA-Ligase, Code no.27-0870-04). The ligation mixture was then packed in phages with the aidof Gigapack II XL Packing Extracts (Stratagene, La Jolla, USA, ProductDescription Gigapack II XL Packing Extract, Code no. 200217). Forinfection of the E. coli strain NM554 (Raleigh et al. 1988, Nucleic AcidResearch 16:1563-1575) the cells were taken up in mM MgSO₄ and mixedwith an aliquot of the phage suspension. The infection and titering ofthe cosmid library were carried out as described by Sambrook et al.(1989, Molecular Cloning: A laboratory Manual, Cold Spring Harbor), thecells being plated out on LB agar (Lennox, 1955, Virology, 1:190) with100 μg/ml ampicillin. After incubation overnight at 37° C., recombinantindividual clones were selected.

Example 2

Isolation and Sequencing of the pfk Gene

The cosmid DNA of an individual colony was isolated with the QiaprepSpin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) inaccordance with the manufacturer's instructions and partly cleaved withthe restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany,Product Description Sau3AI, Product No. 27-0913-02). The DNA fragmentswere dephosphorylated with shrimp alkaline phosphatase (Roche MolecularBiochemicals, Mannheim, Germany, Product Description SAP, Product No.1758250). After separation by gel electrophoresis, the cosmid fragmentsin the size range of 1500 to 2000 bp were isolated with the QiaExII GelExtraction Kit (Product No. 20021, Qiagen, Hilden, Germany). The DNA ofthe sequencing vector pZero-1, obtained from the company Invitrogen(Groningen, The Netherlands, Product Description Zero Background CloningKit, Product No. K2500-01) was cleaved with the restriction enzyme BamHI(Amersham Pharmacia, Freiburg, Germany, Product Description BamHI,Product No. 27-0868-04). The ligation of the cosmid fragments in thesequencing vector pZero-1 was carried out as described by Sambrook etal. (1989, Molecular Cloning: A laboratory Manual, Cold Spring Harbor),the DNA mixture being incubated overnight with T4 ligase (PharmaciaBiotech, Freiburg, Germany). This ligation mixture was thenelectroporated (Tauch et al. 1994, FEMS Microbiol Letters, 123:343-7)into the E. coli strain DH5αMCR (Grant, 1990, Proceedings of theNational Academy of Sciences U.S.A., 87:4645-4649) and plated out on LBagar (Lennox, 1955, Virology, 1:190) with 50 μg/ml zeocin. The plasmidpreparation of the recombinant clones was carried out with Biorobot 9600(Product No. 900200, Qiagen, Hilden, Germany). The sequencing wascarried out by the dideoxy chain termination method of Sanger et al.(1977, Proceedings of the National Academy of Sciences U.S.A.,74:5463-5467) with modifications according to Zimmermann et al. (1990,Nucleic Acids Research, 18:1067). The “RR dRhodamin Terminator CycleSequencing Kit” from PE Applied Biosystems (Product No. 403044,Weiterstadt, Germany) was used. The separation by gel electrophoresisand analysis of the sequencing reaction were carried out in a“Rotiphoresis NF Acrylamide/Bisacrylamide” Gel (29:1) (Product No.A124.1, Roth, Karlsruhe, Germany) with the “ABI Prism 377” sequencerfrom PE Applied Biosystems (Weiterstadt, Germany).

The raw sequence data obtained were then processed using the Stadenprogram package (1986, Nucleic Acids Research, 14:217-231) version 97-0.The individual sequences of the pZerol derivatives were assembled to acontinuous contig. The computer-assisted coding region analysis [sic]were prepared with the XNIP program (Staden, 1986, Nucleic AcidsResearch, 14:217-231). Further analyses were carried out with the “BLASTsearch program” (Altschul et al., 1997, Nucleic Acids Research,25:3389-3402), against the non-redundant databank of the “NationalCenter for Biotechnology Information” (NCBI, Bethesda, Md., USA).

The resulting nucleotide sequence is shown in SEQ ID NO:1. Analysis ofthe nucleotide sequence showed an open reading frame of 990 base pairs,which was designated the pfk gene. The pfk gene encodes a protein of 330amino acids.

Example 3

Preparation of the Expression Vector pZ-pfkex for Enhancement of the pfkGene in Corynebacterium glutamicum

3.1. Cloning of the pfk Gene

From the strain ATCC 13032, chromosomal DNA was isolated by the methodof Eikmanns et al. (Microbiology 140: 1817-1828 (1994)). On the basis ofthe sequence of the pfk gene known for C. glutamicum from example 2, thefollowing oligonucleotides were chosen for the polymerase chainreaction:

pfk-ex: 5′ GAT CTA GAA TTC AAC TTT CAG GTG GTA ACC C 3′

pfk-glp2: 5′ GAT CTA GTC GAC CGG ACA AGC GAG GAA TTA T 3′

The primers described were synthesized by ARK Scientific GmbH Biosystems(Darmstadt, Germany). The primer pfk-ex contains the sequence for thecleavage site of the restriction endonuclease EcoRI and the primerpfk-glp2 the cleavage site of the restriction endonuclease SalI, whichare marked by underlining in the nucleotide sequence shown above. ThePCR reaction was carried out by the standard PCR method of Innis et al.(PCR protocols. A Guide to Methods and Applications, 1990, AcademicPress) with Pwo-Polymerase from Roche Diagnostics GmbH (Mannheim,Germany). With the aid of the polymerase chain reaction, the primersallow amplification of a DNA fragment of approx. 1.05 kb in size, whichcarries the pfk gene from Corynebacterium glutamicum. The productamplified in this way was tested electrophoretically in a 0.8% agarosegel.

The PCR fragment obtained in this manner was cleaved completely with therestriction enzymes EcoRI and SalI. The pfk fragment approx. 1.05 kb insize was isolated from the agarose gel with the QiaExII Gel ExtractionKit (Product No. 20021, Qiagen, Hilden, Germany).

3.2. Cloning of pfk in the Vector pZ8-1

The E. coli-C. glutamicum-shuttle-expression vector pZ8-1 (EP 0 375 889)was employed as the base vector for expression both in C. glutamicum andin E. coli. DNA of this plasmid was cleaved completely with therestriction enzymes EcoRI and SalI and then dephosphorylated with shrimpalkaline phosphatase (Roche Diagnostics GmbH, Mannheim, Germany, ProductDescription SAP, Product No. 1758250). The pfk fragment isolated fromthe agarose gel in example 3.1 was mixed with the vector pZ8-1 preparedin this way and the batch was treated with T4 DNA ligase (AmershamPharmacia, Freiburg, Germany, Product Description T4-DNA-Ligase, Codeno. 27-0870-04).

The ligation batch was transformed in the E. coli strain DH5αmcr(Hanahan, In: DNA cloning. A Practical Approach, Vol. I, IRL-Press,Oxford, Washington D.C., USA). Selection of plasmid-carrying cells wasmade by plating out the transformation batch on LB agar (Lennox, 1955,Virology, 1:190) with 50 mg/l kanamycin. After incubation overnight at37° C., recombinant individual clones were selected. Plasmid DNA wasisolated from a transformant with the Qiaprep Spin Miniprep Kit (ProductNo. 27106, Qiagen, Hilden, Germany) in accordance with themanufacturer's instructions and investigated by restriction cleavage.The resulting plasmid was called pZ-pfkex. It is shown in FIG. 1.

Example 4

Transformation of the Strain DSM5715 with the Plasmid pZ-pfkex

The strain DSM5715 was transformed with the plasmid pZ-pfkex using theelectroporation method described by Liebl et al., (FEMS MicrobiologyLetters, 53:299-303 (1989)). Selection of the transformants took placeon LBHIS agar comprising 18.5 g/l brain-heart infusion broth, 0.5 Msorbitol, 5 g/l Bacto-tryptone, 2.5 g/l Bacto-yeast extract, 5 g/l NaCland 18 g/l Bacto-agar, which had been supplemented with 25 mg/lkanamycin. Incubation was carried out for 2 days at 33° C.

Plasmid DNA was isolated from a transformant by conventional methods(Peters-Wendisch et al., 1998, Microbiology 144, 915-927), cleaved withthe restriction endonucleases EcoRI and SalI, and the plasmid waschecked by subsequent agarose gel electrophoresis. The resulting strainwas called DSM5715/pZ-pfkex.

Example 5

Preparation of Lysine

The C. glutamicum strain DSM5715/pZ-pfkex obtained in example 4 wascultured in a nutrient medium suitable for the production of lysine andthe lysine content in the culture supernatant was determined.

For this, the strain was first incubated on an agar plate with thecorresponding antibiotic (brain-heart agar with tetracycline (5 mg/l))for 24 hours at 33° C. Starting from this agar plate culture, apreculture was seeded (10 ml medium in a 100 ml conical flask). Thecomplete medium Cg III was used as the medium for the preculture. MediumCg III NaCl 2.5 g/l Bacto-Peptone 10 g/l Bacto-Yeast extract 10 g/lGlucose (autoclaved separately) 2% (w/v) The pH was brought to pH 7.4

Kanamycin (25 mg/l) was added to this. The preculture was incubated for16 hours at 33° C. at 240 rpm on a shaking machine. A main culture wasseeded from this preculture such that the initial OD (660 nm) of themain culture was 0.05. Medium MM was used for the main culture. MediumMM CSL (corn steep liquor) 5 g/l MOPS (morpholinopropanesulfonic 20 g/lacid) Glucose (autoclaved separately) 50 g/l (NH₄)₂SO₄ 25 g/l KH₂PO₄ 0.1g/l MgSO₄ * 7 H₂O 1.0 g/l CaCl₂ * 2 H₂O 10 mg/l FeSO₄ * 7 H₂O 10 mg/lMnSO₄ * H₂O 5.0 mg/l Biotin (sterile-filtered) 0.3 mg/l Thiamine * HCl(sterile-filtered) 0.2 mg/l L-Leucine (sterile-filtered) 0.1 g/l CaCO₃25 g/l

The CSL, MOPS and the salt solution were brought to pH 7 with aqueousammonia and autoclaved. The sterile substrate and vitamin solutions werethen added, as well as the CaCO₃ autoclaved in the dry state.

Culturing is carried out in a 10 ml volume in a 100 ml conical flaskwith baffles. Kanamycin (25 mg/l) was added. Culturing was carried outat 33° C. and 80% atmospheric humidity.

After 72 hours, the OD was determined at a measurement wavelength of 660nm with a Biomek 1000 (Beckmann Instruments GmbH, Munich). The amount oflysine formed was determined with an amino acid analyzer fromEppendorf-BioTronik (Hamburg, Germany) by ion exchange chromatographyand post-column derivatization with ninhydrin detection.

The result of the experiment is shown in table 1. TABLE 1 Strain OD(660)Lysine HCl g/l DSM5715 7.9 13.0 DSM5715/pZ-pfkex 9.9 13.4

1-20. (canceled)
 21. A process for the production of an L-amino acidcomprising the following steps: a) culturing a coryneform bacteriumcomprising an overexpressed pfk gene, wherein said pfk gene comprises apolynucleotide having a nucleotide sequence which is at least 90%identical to the nucleotide sequence of SEQ ID NO: 1 encoding apolypeptide having the enzymatic activity of a phosphofructokinase, andcomprising one or more overexpressed genes selected from the groupconsisting of the dapA gene of Corynebacterium glutamicum encodingdihydrodipicolinate synthase, the pyc gene of Corynebacterium glutamicumencoding pyruvate carboxylase, the tpi gene of Corynebacteriumglutamicum encoding triose phosphate isomerase, the gap gene ofCorynebacterium glutamicum encoding glyceraldehyde 3-phosphatedehydrogenase and the pgk gene of Corynebacterium glutamicum encoding3-phosphoglycerate kinase, b) accumulating said L-amino acid in themedium or in the cells of said bacterium c) isolating said L-amino acid.22. The process of claim 1, wherein said polypeptide having theenzymatic activity of a phosphofructokinase comprises the amino acidsequence of SEQ ID NO:
 2. 23. The process of claim 1, wherein saidpolynucleotide comprises the nucleotide sequence of nucleotides 68 to1057 of SEQ ID NO:
 1. 24. The process of claim 1, wherein saidpolynucleotide comprises the nucleotide of SEQ ID NO:
 1. 25. The processof claim 1, wherein said overexpression is achieved by increasing thecopy number of said pfk gene or by operably linking said pfk gene to apromoter.